Enhanced expression of endogenous retroviruses and of TRIM28 and SETDB1 in children with food allergy

Abstract Background Human endogenous retroviruses (HERVs) represent 8% of our genome. They originate from ancestral infections and although no longer contagious they can regulate transcription of adjacent cellular genes, produce viral RNAs sensed as non‐self by pattern recognition receptors, and encode viral proteins, such as Syncytin (SYN) 1 and 2, that exhibit potent immunomodulatory properties. Based on this, HERVs have been studied and proposed as relevant cofactors in several chronic inflammatory and immune‐mediated diseases. HERV transcription is regulated by host TRIM28 and SET domain bifurcated histone lysine methyltransferase 1 (SETDB1), which in turn exert crucial regulatory functions on the host immune system. No studies explored the expression of HERVs, TRIM28, and SETDB1 in allergic patients. Methods We assessed, through a polymerase chain reaction real time Taqman amplification assay, the transcription levels of pol genes of HERV‐H, HERV‐K, HERV‐W, and of env genes of SYN1 and SYN2, as well as of TRIM28 and SETDB1 in whole blood from 32 children with IgE‐mediated food allergy, 19 with food protein‐induced enterocolitis syndrome (FPIES), and in healthy control children. Results The expression levels of pol genes of HERV‐H, ‐K, and ‐W were significantly enhanced in patients with IgE‐mediated FA or FPIES as compared to control subjects, while the mRNA concentrations of SYN1 and SYN2 were comparable in each group of children. Both TRIM28 and SETDB1 mRNA levels were significantly higher in allergic patients. Conclusions Given the influence of HERVs and of TRIM28 and SETDB1 on innate and adaptive immune responses, their transcriptional activation in children with food allergies suggest that they might play important roles in the development of these diseases.


S C H L Ü S S E L W Ö R T E R
Endogene retroviren, Kinder, Lebensmittelallergie, SETDB1, TRIM28

| INTRODUCTION
Allergic diseases are worldwide among the most common chronic inflammatory disorders, especially in pediatric age. They represent an abnormal reaction to an ordinarily harmless substance referred to as allergen. Food allergy (FA) is an immune-mediated adverse reaction to specific food(s) with protean clinical manifestations, that is thought to be triggered by a combination of genetic and environmental factors. 1 It encompasses IgE-driven and non-IgE-mediated food-induced allergic disorders, such as the food protein-induced enterocolitis syndrome (FPIES). 2 A key role for the immunological tolerance to foods is thought to be played by dendritic cells (DCs) in the gastrointestinal tract and DCs plus Langerhans cells in the skin, which direct the response of regulatory T cells (Tregs) towards a tolerogenic pattern. 3,4 In patients with IgE-mediated FA, the induction of Tregs is altered and it is replaced by a Th2 response leading to the synthesis of food antigenspecific IgE antibodies. 3

IL-33-driven induction of large amounts of IL-4 and IgE-mediated activation of mast cells further contribute to
Treg skewing and the amplification of Th2 response. 5 The immunopathology of FPIES remains elusive. 6 The syndrome is regarded as a cell-mediated immune disorder, with food allergens triggering T cell activation that results in abnormal release of proinflammatory cytokines, ultimately leading to intestinal damage. 7 Recent studies suggest that the innate immune response plays a key role with production of a large array of chemokines and cytokines as well as increased expression of neutrophil and monocyte activation genes. 7,8 Human endogenous retroviruses (HERVs) originate from ancestral infections that led to their integration into the genome of primates more than 25 millions of years ago. 9 During evolution, the accumulation of mutations blocked the production of infectious virions and most HERVs became inactive. However, some viral sequences are transcribed and a few encode proteins, such as the Syncytin 1 (SYN1) 10 and Syncytin 2 (SYN2), 11 that play crucial roles in placenta morphogenesis and in feto-maternal tolerance. 12 HERV elements contribute to the regulation of essential immune functions. They are extensively distributed throughout the human genome and can modulate transcription of close cellular genes. 13,14 Their RNAs, through retrotransposition, can generate novel insertions into the genome and, being sensed as non-self by pattern recognition receptors (PRRs), they can elicit inflammatory and immune reactions. [13][14][15][16] Furthermore, some viral proteins can trigger autoimmunity, 17,18 while others, such as the syncytins, exhibit intrinsic immunomodulatory properties. 12,19,20 Several lines of research have evidenced an association between aberrant HERV expressions and immune-mediated diseases, supporting the etiopathogenetic role of retroviruses in these pathologies. 18,[21][22][23] Activation of HERVs may be regulated by environmental factors via epigenetic mechanisms, such as DNA methylation and heterochromatin-silencing by histone modifications. Krüppelassociated box domain zinc finger proteins (KRAB-ZFPs) are the largest family of transcriptional regulators in the human genome. 24 Tripartite motif containing 28 (TRIM28), also called KAP1 or TIF1-β, is a nuclear co-repressor of KRAB-ZFPs. 25 SET domain bifurcated histone lysine methyltransferase 1 (SETDB1), also known as ERGassociated Protein with a SET domain, is a methyltransferase with high specificity for the lysine 9 residue of histone H3. 26 Both TRIM28 and SETBD1 represent specific tags for epigenetic transcriptional repression of sequences derived from HERV elements. 27,28 Additionally, growing data document their involvement in many aspects of cell homeostasis and in epigenetic control of both innate and adaptive immune responses. 29,30 Notably, accumulating evidence highlights the importance of epigenetic mechanisms in the pathogenesis of allergic diseases, 31,32 including FA. 33,34 The understanding of underlying pathophysiology and the identification of molecules involved in the development of FA may contribute to its better prevention and to innovative treatment modalities. 35 Despite the potential impact of HERVs and of TRIM28 and SETDB1 in triggering and/or maintaining allergic reactions, to the best of our knowledge no studies explored their expressions in allergic patients. The aims of the current study were to assess the transcription levels of pol genes of HERV-H, -K, and -W, the three retroviral families most widely studied, 9,21 of env genes of SYN1 and SYN2 as well as of TRIM28 and SETDB1 in whole blood from children affected by IgE-mediated FA or FPIES and in control healthy children.

| Study populations
Three groups of children were investigated: subjects with IgEmediated FA (Group A), subjects with FPIES (Group B), and control healthy subjects (Group C).
Group A and Group B children were enrolled at Pediatric Allergy Unit of the Regina Margherita Children's Hospital, Turin, Italy. They were tested after suspension from at least 1-3 months of the triggering food(s) during a routine laboratory control. At time of testing, all subjects were in good general condition, had no symptoms or signs of allergy, and inflammatory markers (WBC count, C reactive protein, erythrocyte sedimentation rate) were all within the normal range.
The Group C included asymptomatic children who were tested at the Regina Margherita Children's Hospital for routine laboratory examinations and whose results were all within the normal reference range. Subjects with any confirmed or suspected disease, such as allergy, infections, cancer, autoimmune disorders, inflammatory diseases, neurological disturbances, or abnormal laboratory results were excluded from the study.

| Diagnostic criteria
The diagnosis of IgE-mediated FA was based on the personal history of acute allergic symptoms (cutaneous, ocular, upper/lower respiratory, oral/lower gastrointestinal, cardiovascular, anaphylaxis) appearing within 1 h after ingestion of a specific food and confirmed by oral food challenge (except for severe anaphylaxis), a positive skin prick test and/or a serum food-specific IgE level of ≥ 0.10 kU/l. When available, sIgE antibodies against food molecules were also investigated (component-resolved diagnostic tests [CRD]). 36,37 FPIES was diagnosed according to the international consensus guidelines for its diagnosis and management. 38

| Total RNA extraction
Total RNA was extracted from whole blood using the automated   The amplifications were run in a 96-well plate at 95°C for 10 min, followed by 45 cycles at 95°C for 15 s and at 60°C for 1 min. Each sample was run in triplicate. RQ of target gene transcripts was performed with the ΔΔCt method. Hence, fold change was calculated and results were expressed in corresponding arbitrary units, called RQ. Since we measured Ct for every target in all samples, we argued that our methods were suitable for HERV detection and quantification.

| Statistical analysis
One-way analysis of variance (ANOVA) test was used to compare the transcriptional levels of pol genes of HERV-H, -K, and -W, of env genes of SYN1, SYN2, and of TRIM28 and SETDB1 between the three groups of children. Mann-Whitney test was used to compare the transcripts of pol genes of every HERV family as well as of SYN1, SYN2, TRIM28, and SETDB1 between each group of children. Mann-Whitney test was used to compare the transcription levels of HERVs between males and females. Spearman correlation test was used to evaluate the correlations between age and the transcription levels of each HERV sequence and of TRIM28 and SETDB1. Statistical analyses were done using the Prism software (GraphPad Software). In all analyses, p < 0.05 was taken to be statistically significant. TOVO ET AL.

| Influence of age and gender on transcription levels of HERVs, TRIM28, and SETDB1
The median age was significantly different in the three groups of children. In particular, patients with IgE-mediated FA were older than children of the control groups (p < 0.0001 for Group C1; p < 0.0001 for Group C2; p = 0.0011 for Group C3) and of patients with FPIES (p = 0.0007). These were younger than children of C1 and C3 control groups (p = 0.0349 and p = 0.0011, respectively) while their age was comparable to C2 children (p = 0.1398).
The transcriptional levels of each target gene were however not related to the age. As illustrated in Figure 1, no significant correla-

| Transcription levels of HERV-H-pol, HERV-Kpol, HERV-W-pol, SYN1-env, and SYN2-env in children with FA and control children
The ANOVA analysis showed that there was a statistically sig- In contrast, as illustrated in Figure 4, no significant differences were observed in the mRNA concentrations of SYN1-env and SYN2env between the three groups of children. .

| Transcription levels of TRIM28 and SETDB1 in children with FA and control children
The ANOVA analysis showed that there were statistically significant differences in the transcription levels of TRIM28 and SETDB1 between the three groups of subjects ( This accounts for the younger age of these patients as compared to control subjects or patients with IgE-mediated FA. However, in control children the age did not influence the expression of any target gene and similar findings were observed in allergic patients.
Therefore, the impact of the age on the trans-activation of the genes here analyzed was irrelevant. No significant differences emerged also between males and females, in line with lack of association between sex and FA. 45 In contrast to HERV-pol sequences, both SYN1-env and SYN2env mRNA levels were comparable in the three groups of subjects.
There is a general agreement that syncytins exert important regulatory functions on a large array of immune responses and in the induction of immune-tolerance. 12,14,19,20 However, the normal expression profiles of SYN1 and SYN2 in children with IgE-mediated FA or FPIES suggest that they do not play an important role in the pathogenesis of these diseases. Their higher expressions result in enhanced DNA methylation and heterochromatin formation ultimately leading to HERV silencing. 27,28 However, TRIM28 and SETDB1 mRNA concentrations were unexpectedly not reduced, but enhanced in both groups of allergic patients. Therefore, the enhanced HERV-pol activation cannot be ascribed to impaired transcription of TRIM28 or SETDB1 repressors.
It is worth noting that no reduced TRIM28/SETDB1 expression was found also in other immune-mediated diseases characterized by increased levels of HERV-pol mRNAs, such as in new-onset type 1 diabetes 22 and celiac disease. 23 Allergy is thought to originate from a dysfunction of the immune system. As mentioned, growing evidence indicates that endogenous retroviruses can condition and shape the host immune response.
There is wide consensus on the crucial role played by forkhead box p3+ (Foxp3) Tregs to ensure immunological tolerance, while their dysfunctions may lead to allergic reactions, 46,47 including FA. 48 In mice, retroviral superantigens have been shown to induce expansion and activation of Foxp3+ Tregs. 49 This notwithstanding, whether HERVs are really responsible for immune-mediated damages or their activation represents a secondary epiphenomenon remains an unsolved dilemma. Inflammatory-driven activation of NF-kB and production of pro-inflammatory cytokines can elicit HERV transcription. 50 On the other hand, HERVs can in turn induce flogosis, 9,16,21 giving rise to a vicious circle to trigger local and systemic inflammatory responses. In this context, it must be underlined that in our patients HERV upregulation was detected after suspension of the Our results on HERV-pol expressions have some technical limits which did not allow to better characterize the potential involvement of HERV copies with transcriptional activity, in particular the contribution of specific HERV loci. Furthermore, we did not assess their protein-coding capacity.

DECLARATIONS
The study was carried out in accordance with the principles of Hel-

ACKNOWLEDGMENT
This research received no external funding.

CONFLICT OF INTEREST
The authors declare no conflict of interest.